Antiviral Effect Of Recombinant IFNα2b On PHEV-infected N2a Cells And Related Mechanisms

Porcine hemagglutinating encephalomyelitis virus(PHEV)was isolated and identified in 1962 and belongs to porcine β coronavirus.As a neurotropic virus,it spreads peripherally to the central nervous system(CNS),causing nerve damage.It can cause piglet encephalomyelitis,vomiting and wasting diseases,seriously damaging the pig industry.In the absence of effective therapeutic drugs and vaccines,strengthening integrated prevention and control is the ultimate strategy to treat the disease.Interferon is a class of multifunctional and bioactive proteins produced by human or animal bodies after being stimulated by viruses and other substances.The biological activity of type I interferon(IFN-I)is mainly antiviral,IFN-I has three types: α,β and γ,among which α and β have broad-spectrum antiviral biological effects,which have been widely used in the prevention and treatment of viral infectious diseases.Interferons in the domestic market are produced using recombinant gene technology.At present,type α interferon is the most widely used in clinical practice.There are three types of interferon type α,α1b,α2a,α2b,which can achieve efficient broad-spectrum antiviral effects.so we combined with a new nanogel interferon α2b developed by a biomedical company to study the antiviral effect and related mechanism of PHEV.In order to clarify the anti-PHEV effect of IFNα2b and find new antiviral targets and molecular mechanisms,this study used mouse neuroma blasts(Neuro-2a,N2a)as a cell model to study IFNα2b anti-PHEV.The IFNα2b use concentration and the viral titers of PHEV were first determined.The optimal time for viral infection and drug action is determined by Western Blot.By first adding IFNα2b and then inoculating the virus,and inoculating the virus first and then adding IFNα2b and adding both virus and IFNα2b to treat the cells.Western Blot,q RT-PCR and immunofluorescence methods were used to detect the antiviral effect of IFNα2b,the results show that IFNα2b could significantly reduce the content of the virus in cells,The trends in m RNA levels and protein levels of the three specific molecules Mx1,OAS1 a and ISG15 specifically induced by interferon indicated that IFNα2b had significant antiviral replication effects.Studies have shown that PHEV replication and proliferation within host cells are associated with autophagy,and IFNα2b will also exert its antiviral effect by regulating autophagy.In order to explore the relationship between PHEV replication and autophagy,whether IFNα2b anti-PHEV is related to autophagy.In this study,Western Blot,and double fluorescent m RFP-GFP-LC3 experiment were applied.The results showed that PHEV induced incomplete autophagy in N2 a cells in a non-acidifying lysosomal manner,and evaded degradation by autophagic lysosomes by inhibiting the fusion of autophagosomes and lysosomes.Autophagosomes may be involved in PHEV replication.IFNα2b can regulate autophagy flow in N2 a cells after PHEV infection,promote the fusion of autophagosomes and lysosomes to inhibit the proliferation and replication of PHEVs.In conclusion,this study revealed that recombinant IFNα2b can inhibit the replication and proliferation of PHEV viruses,and initially revealed that IFNα2b exerts antiviral effects by regulating the development of autophagy.The results provide a basis for finding new antiviral targets and molecular mechanism of IFNα2b.